QPCTL (i.e. glutaminyl peptide cyclotransferase like), also termed Iso-glutaminyl cyclase (isoQC) (see SEQ ID NO's: 2, 5 and 7 for the QPCTL's from mouse, rat and human, respectively and SEQ ID NO's: 1, 4 and 6 for the cDNA sequences of the QPCTL's from mouse, rat and human, respectively) catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid (5-oxo-proline, pGlu*) with liberation of ammonia and the intramolecular cyclization of N-terminal glutamate residues into pyroglutamic acid with liberation of water.
Glutaminyl cyclase (QC, EC 2.3.2.5) (or QPCT) catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid (pGlu*) liberating ammonia. A QC was first isolated by Messer from the Latex of the tropical plant Carica papaya in 1963 (Messer, M. (1963) Nature 4874, 1299). 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. (1987) J Biol. Chem. 262, 8532-8536; Fischer, W. H. and Spiess, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3628-3632). For the mammalian QC, the conversion of Gln into pGlu by QC could be shown for the precursors of TRH and GnRH (Busby, W. H. J. et al. (1987) J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. (1987) Proc Natl Acad Sci U.S.A. 84, 3628-3632). In addition, initial localization experiments of QC revealed a co-localization with its putative products of catalysis in bovine pituitary, further improving the suggested function in peptide hormone synthesis (Bockers, T. M. et al. (1995) J Neuroendocrinol. 7, 445-453). In contrast, the physiological function of the plant QC is less clear. In case of the enzyme from C. papaya, a role in the plant defence against pathogenic microorganisms was suggested (El Moussaoui, A. et al. (2001) Cell. Mol. Life. Sci. 58, 556-570). Putative QCs from other plants were identified by sequence comparisons (Dahl, S. W. et al. (2000) Protein Expr. Purif. 20, 27-36). The physiological function of these enzymes, however, is still ambiguous.
The QCs known from plants and animals show a strict specificity for L-glutamine in the N-terminal position of the substrates and their kinetic behavior was found to obey the Michaelis-Menten equation (Pohl, T. et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10059-10063; Consalvo, A. P. et al. (1988) Anal. Biochem. 175, 131-138; Gololobov, M. Y. et al. (1996) Biol. Chem. Hoppe Seyler 377, 395-398). A comparison of the primary structures of the QCs from C. papaya and that of the highly conserved QC from mammals, however, did not reveal any sequence homology (Dahl, S. W. et al. (2000) Protein Expr. Purif. 20, 27-36). Whereas the plant QCs appear to belong to a new enzyme family (Dahl, S. W. et al. (2000) Protein Expr. Purif. 20, 27-36), the mammalian QCs were found to have a pronounced sequence homology to bacterial aminopeptidases (Bateman, R. C. et al. (2001) Biochemistry 40, 11246-11250), leading to the conclusion that the QCs from plants and animals have different evolutionary origins.
The subject matter of the present invention is particularly useful in the field of QPCT-related diseases, one example of those being Alzheimer's Disease, whereby these diseases are simultaneously QPCTL-related in view of the above-described similarly catalyzed reaction. Alzheimer's disease (AD) is characterized by abnormal accumulation of extracellular amyloidotic plaques closely associated with dystrophic neurones, reactive astrocytes and microglia (Terry, R. D. and Katzman, R. 1983 Ann. Neurol. 14, 497-506; Glenner, G. G. and Wong, C. W. (1984) Biochem. Biophys. Res. Comm. 120, 885-890; Intagaki, S. et al. (1989) J Neuroimmunol. 24, 173-182; Funato, H. et al. (1998) Am. J Pathol. 152, 983-992; Selkoe, D. J. (2001) Physiol. Rev. 81, 741-766). Amyloid-beta (abbreviated as Aβ) peptides are the primary components of senile plaques and are considered to be directly involved in the pathogenesis and progression of AD, a hypothesis supported by genetic studies (Glenner, G. G. and Wong, C. W. (1984) Biochem. Biophys. Res. Comm. 120, 885-890; Borchelt, D. R. et al. (1996) Neuron 17, 1005-1013; Lemere, C. A. et al. (1996) Nat. Med. 2, 1146-1150; Mann, D. M. and Iwatsubo, T. (1996) Neurodegeneration 5, 115-120; Citron, M. et al. (1997) Nat. Med. 3, 67-72; Selkoe, D. J. (2001) Physiol. Rev. 81, 741-766). Aβ is generated by proteolytic processing of the β-amyloid precursor protein (APP) (Kang, J. et al. (1987) Nature 325, 733-736; Selkoe, D. J. (1998) Trends Cell. Biol. 8, 447-453), which is sequentially cleaved by β-secretase at the N-terminus and by γ-secretase at the C-terminus of Aβ (Haass, C. and Selkoe, D. J. (1993) Cell 75, 1039-1042; Simons, M. et al. (1996) J Neurosci. 16 899-908). In addition to the dominant Aβ peptides starting with L-Asp at the N-terminus (Aβ1-42/40), a great heterogeneity of N-terminally truncated forms occurs in senile plaques. Such shortened peptides are reported to be more neurotoxic in vitro and to aggregate more rapidly than the full-length isoforms (Pike, C. J. et al. (1995) J Biol. Chem. 270, 23895-23898). N-truncated peptides are known to be overproduced in early onset familial AD (FAD) subjects (Saido, T. C. et al. (1995) Neuron 14, 457-466; Russo, C, et al. (2000) Nature 405, 531-532), to appear early and to increase with age in Down's syndrome (DS) brains (Russo, C. et al. (1997) FEBS Lett. 409, 411-416, Russo, C. et al. (2001) Neurobiol. Dis. 8, 173-180; Tekirian, T. L. et al. (1998) J Neuropathol. Exp. Neurol. 57, 76-94). Finally, their amount reflects the progressive severity of the disease (Russo, C. et al. (1997) FEBS Lett 409, 411-416; Güntert, A. et al. (2006) Neuroscience 143, 461-475). Additional post-translational processes may further modify the N-terminus by isomerization or racemization of the aspartate at position 1 and 7 and by cyclization of glutamate at residues 3 and 11. Pyroglutamate-containing isoforms at position 3 [pGlu3Aβ3-40/42] represent the prominent forms—approximately 50% of the total Aβ amount—of the N-truncated species in senile plaques (Mori, H. et al. (1992) J Biol. Chem. 267, 17082-17086, Saido, T. C. et al. (1995) Neuron 14, 457-466; Russo, C. et al. (1997) FEBS Lett. 409, 411-416; Tekirian, T. L. et al. (1998) J Neuropathol Exp Neurol 57, 76-94; Geddes, J. W. et al. (1999) Neurobiol Aging 20, 75-79; Harigaya, Y. et al. (2000) Biochem. Biophys. Res. Commun. 276, 422-427) and they are also present in pre-amyloid lesions (Lalowski, M. et al. (1996) J Biol. Chem. 271, 33623-33631). The accumulation of AβN3 (pE) peptides is likely due to the structural modification that enhances aggregation and confers resistance to most amino-peptidases (Saido, T. C. et al. (1995) Neuron 14, 457-466; Tekirian, T. L. et al. (1999) J Neurochem 73, 1584-1589). This evidence provides clues for a pivotal rote of AβN3 (pE) peptides in AD pathogenesis. However, relatively little is known about their neurotoxicity and aggregation properties (He, W. and Barrow, C. J. (1999) Biochemistry 38, 10871-10877; Tekirian, T. L. et al. (1999) J Neurochem. 73, 1584-1589). Moreover, the action of these isoforms on glial cells and the glial response to these peptides are completely unknown, although activated glia is strictly associated with senile plaques and might actively contribute to the accumulation of amyloid deposits. In recent studies the toxicity, aggregation properties and catabolism of Aβ1-42, Aβ1-40, [pGlu3]Aβ3-42, [pGlu3]Aβ3-40, [pGlu11]Aβ11-42 and [pGlu11]Aβ11-40 peptides were investigated in neuronal and glial cell cultures, and it was shown that pyroglutamate modification exacerbates the toxic properties of Aβ-peptides and also inhibits their degradation by cultured astrocytes. Shirotani et al. investigated the generation of [pGlu3]Aβ peptides in primary cortical neurons infected by recombinant Sindbis virus in vitro. They constructed amyloid precursor protein complementary DNAs, which encoded a potential precursor for [pGlu3]Aβ by amino acid substitution and deletion. For one artificial precursor starting with an N-terminal glutamine residue instead of glutamate in the natural precursor, a spontaneous conversion or an enzymatic conversion by glutaminyl cyclase to pyroglutamate was suggested. The cyclization mechanism of N-terminal glutamate at position 3 in the natural precursor of [pGlu3]Aβ was neither determined in vitro, in situ nor in vivo (Shirotani, K. et al. (2002) NeuroSci. Lett. 327, 25-28).
Thus, there is a clear need in the art for the provision of knock-out animals, in particular knock-out mice which carry a knock-out mutation in the QPCTL gene, to enable exact investigations as to the function, relevance and potential of the QPCTL gene as well as the QPCTL protein.
The aim of this invention was to develop knock-out animals, i.e. mouse models carrying a constitutive mutation of the QPCTL gene.